In 1934, a doctor in Norway named Asbjorn Fölling noticed that several mentally retarded patients had a strange odor. He figured out that it was from something called “phenylacetic acid”. The patients' urine also had a very high level of a chemical called “phenylketone”, which is reflected in the name of the disease, phenylketon-uria. Fölling also thought the disease was most likely inherited, and was the first to suggest using diet to manage it. Since then, giant steps have been made in understanding and treating PKU.
Phenylketonuria (PKU), one of the most common inborn error of amino acid metabolism, results from an impaired ability to metabolize the essential amino acid phenylalanine (Phe)[1] and convert it to its hydroxylated derivative tyrosine (Tyr). Classical PKU is a rare metabolic disorder and classified as an orphan disease both according to US and European definitions. PKUusually results from a deficiency of a liver enzyme known as phenylalanine hydroxylase (PAH) but can also result from deficiencies in enzymes needed to produce pyridoxalphosphate, an obligate co-factor to PAH. The enzyme deficiency leads to accumulation of Phe in the blood and other tissues. Phenylalanine is found in breast milk, many types of baby formula, and most foods, especially those containing a high concentration of protein, such as meat, eggs, and dairy products. If PKU is not treated, phenylalanine will build up in the blood and eventually lead to irreversible intellectual disability and problems within the central nervous system (brain and spinal cord). The untreated state is characterized by mental retardation, microcephaly, delayed speech, seizures, eczema, behavioral abnormalities, and other symptoms. The good news is that early treatment can prevent all or most problems. Babies born with PKU need to start treatment with special formula soon after birth. The mainstream treatment for classic PKU patients is a strict Phe-restricted diet supplemented by a medical formula containing free amino acids except Phe and other nutrients covering the demands of the organism of essential amino acids. In the United States, the current recommendation is that the PKU diet should be maintained for life. Patients who are diagnosed early and maintain a strict diet can have a normal life span with normal mental development.
Compliance to a strict Phe-restricted diet supplemented with a medical formula will, however, decrease as the subject gets older and this decrease in compliance may cause later development of cognitive dysfunction. Inadequate compliance may be due to the unpleasant taste and smell of the amino acids formulations as well as a desire to live like normal subjects, who do not suffer from PKU.
The present invention provides recombinant Phe-free proteins for use in the treatment of PKU. Such proteins may be used as such or they may be incorporated into foods. The recombinant Phe-free proteins according to the invention have advantages over the known medical food proteins used in the treatment of PKU as they have improved properties with respect to i) taste, ii) smell, iii) palatability, and iv) texture and, which enhances the acceptability and compliance of the proteins of the present invention and the compositions/medical food containing them.
In the following is given an overview of the disease and treatment options.
Incidence and Newborn Screening
The incidence of PKU is approximately one in every 15,000 births (1/15,000). It affects around 700,000 people around the globe[5]. The overall birth prevalence of PKU in European, Chinese and Korean populations is ˜1/10,000. The mean incidence of PKU varies widely in different human populations (Table 1). United States Caucasians are affected at a rate of 1 in 10,000. Turkey has the highest documented rate in the world, with 1 in 2,600 births, while countries such as Finland and Japan have extremely low rates with fewer than one case of PKU in 100,000 births. A 1987 study from Slovakia reports a Roma population with an extremely high incidence of PKU (one case in 40 births) due toa frequency of cousin marriages.
PKU is commonly included in the newborn screening panel of most countries, with varied detection techniques. Most babies in developed countries are screened for PKU soon after birth. Screening for PKU is done with bacterial inhibition assay (Guthrie test), immunoassays using fluorometric or photometric detection, or amino acid measurement using tandem mass spectrometry (MS/MS). Measurements done using MS/MS determine the concentration of Phe and the ratio of Phe to Tyr.
If a child is not screened during the routine newborn screening test (typically performed 2-7 days after birth, using samples drawn by neonatal heel prick), the disease may present clinically with seizures, albinism (excessively fair hair and skin), and a “musty odor” to the baby's sweat and urine. In most cases, a repeat test should be done at approximately two weeks of age to verify the initial test and uncover any phenylketonuria that was initially missed. The affected children who are detected and treated are less likely to develop neurological problems or have seizures and mental retardation, though such clinical disorders are still possible.
Phe Metabolic Pathways
Phe exists as D and L enantiomers, and L-Phe is an essential amino acid required for protein synthesis in human[6]. There are many processes (FIG. 1), which contribute to the flux of L-Phe in human. The main pathway is the enzyme PAH normally converts the amino acid phenylalanine into the amino acid tyrosine. However, other pathway reactions also occur in normal liver tissue but are of minor significance. As with many other metabolites, Phe concentrations are regulated to a steady state level with dynamic input and run-out flux. Persistent disturbance to the flux will eventually result in alteration of the steady state concentrations and accumulation of Phe[7]. Excessive Phe can be metabolized into phenylketones through the minor route, a transaminase pathway with glutamate. Metabolites include phenylacetate, phenylpyruvate and phenethylamine. Elevated levels of phenylalanine in the blood and detection of phenylketones in the urine are ways to diagnose PKU, however most patients are diagnosed via newborn screening.
PAH enzyme requires tetrahydrobiopterin (BH4) as an essential co-factor, which is formed in three steps. During the hydroxylation reaction BH4 is converted to the inactive pterin, BH2, dihydrobiopterin (quinone). The enzyme dihydropteridine reductase (DHPR) regenerate BH4 (FIG. 2). BH4 is also an obligate co-factor for tyrosine hydroxylase, tryptophan hydroxylase, and for nitric oxide synthase and is thus necessary for the production of dopamine, catecholamines, melanin, serotonin, and for nitric oxide. Defects in either PAH or the production or recycling of BH4 may result in hyperphenylalaninaemia.
Phenylalanine is a large neutral amino acid (LNAA). LNAAs compete for transport across the blood-brain barrier (BBB) via the large neutral amino acid transporter (LNAAT). If phenylalanine is in excess in the blood, it will saturate the transporter. Excessive levels of phenylalanine tend to decrease the intratechal levels of other LNAAs (Tyr, Trp, Thr, Ile, Leu, Val, Met and His) in the brain. As these amino acids, especially Tyr and Trp, are necessary for protein and neurotransmitter synthesis, Phe buildup hinders the development of the brain, causing mental retardation.
Phenylalanine levels are monitored typically twice a week in neonates, weekly in infants, biweekly or every 3 weeks in toddlers, and monthly thereafter, even during adult life. Attention should be given to variability in blood phenylalanine levels and to maintenance within the recommended range. During pregnancy, weekly phenylalanine sampling is recommended.
Types of PKU
Classical PKU is caused by a mutated gene for the enzyme PAH, which converts the Phe to other essential compounds in the body. Other non-PAH mutations can also cause PKU. The PAH gene is located on chromosome 12 in the bands 12q22-q24.1. More than 400 disease-causing mutations have been found in the PAH gene. PAH deficiency causes a spectrum of disorders, including classic PKU and hyperphenylalaninemia (a less severe accumulation of phenylalanine).
PKU is known to be an autosomal recessive genetic disorder. This means both parents must have at least one mutated allele of the PAH gene. The child must inherit both mutated alleles, one from each parent. Therefore, it is possible for a parent with the disease to have a child without it if the other parent possesses one functional allele of the gene for PAH. Yet, a child from two parents with PKU will inherit two mutated alleles every time and therefore the disease.
PKU can exist in mice, which have been extensively used in experiments into finding an effective treatment for it. The availability of a mutant mouse that closely mimics the human disease, called PAHenu2 provides an ideal model for investigating gene transfer in vivo and invaluable information on the pathology and biology of PKU. Numerous genetic and biochemistry studies have confirmed the reliability of this mouse model to closely resemble the metabolic and neurobiological phenotype of human PKU. The macaque monkey's genome was recently sequenced, and the gene encoding phenylalanine hydroxylase was found to have the same sequence that, in human, would be considered as a PKU mutation.
Tetrahydrobiopterin-deficient hyperphenylalaninemia is a rare, explaining about 1-5% of all PKU cases. These patients have normal PAH, but lack the ability in the biosynthesis or recycling of the cofactor tetrahydrobiopterin (BH4). BH4 is necessary for proper activity of the enzyme. Tetrahydrobiopterin deficiency can be caused by defects in four different genes. These types are known as HPABH4A, HPABH4B, HPABH4C, and HPABH4D.
Treatment of PKU
The foundation of PKU treatment is a low Phe diet which prevents the development of the neurological and psychological changes. Since neurological changes have been demonstrated within one month of birth, it is recommended that dietary restriction should be started early and be continued through childhood when neural development is maximal. Clinical neurological abnormalities, affected neuropsychological performance and brain imaging in adults with PKU has led to a consensus opinion that the PKU diet should be followed for life. An even more stringent regime of Phe restriction is required for women with PKU contemplating starting a family, particularly during pregnancy, as elevated blood Phe concentrations are teratogenic towards the developing foetus. A preconception diet is required with a S-Phe target interval of between 100 and 360 μmol/L in affected mothers.
A Phe-restriction diet can lower plasma Phe levels and may prevent the mental impairments of PKU patients. However, compliance with dietary treatment erodes, as patients get older. Because some patients are not able to adhere rigorously to the phenylalanine-restricted diet during life, alternative treatment regimens have been developed. Moreover, Pregnant PKU/HPA women have a particular need for keeping the Phe levels low, since high level of Phe affects the embryo and fetus (maternal PKU). The UK MRC Study Group on PKU has concluded that there is a need for an alternative to the low-Phe diet. The NIH Consensus Panel also encouraged research on therapeutics for PKU, including enzyme therapy and gene therapy. FIG. 3 shows several kinds of treatment options available in practice or in theory for PKU therapy[8].
Dietary Modifications
If PKU is diagnosed early enough, an affected newborn can grow up with normal brain development, but only by managing and controlling Phe levels through diet, or a combination of diet and medication. An optimal health range of Phe in plasma is between 120 and 360 μmol/L, and a people with PKU should control their Phe for life, as determined by experts convened by the National Institutes of Health (NIH). Most natural foods contain protein containing 2.4-9% Phe by weight[9]. All PKU patients must adhere to a special diet low in Phe for optimal brain development (below 500 mg/day). The diet requires severely restricting or eliminating foods high in Phe, such as meat, chicken, fish, eggs, nuts, cheese, legumes, milk and other dairy products. Starchy foods, such as potatoes, bread, pasta, and corn, must be monitored. Infants may still be breastfed to provide all of the benefits of breast milk, but the quantity must also be monitored and supplementation for missing nutrients will be required. The sweetener aspartame (L-aspartyl-L-Phe methyl ester), present in many diet foods and soft drinks, must also be avoided, as the metabolism of the dipeptide aspartame will release Phe, L-aspartic acid and methanol.
Low-Phe diet often includes: Low-Phe natural foods (some fruits and vegetables), Low-protein specialty foods (low-protein pasta, bread, etc.), Phe-free formula and Phe-free protein replacement bars, tablets, capsules, etc. Supplementary infant formulas are used in these patients to provide the amino acids and other necessary nutrients that would otherwise be lacking in a low-phenylalanine diet. As the child grows up these can be replaced with pills, formulas, and specially formulated foods. Since Phe is necessary for the synthesis of many proteins, it is required for appropriate growth, but levels must be strictly controlled in PKU patients. In addition, tyrosine, which is normally derived from phenylalanine, must be supplemented.
Supplementation with amino acid (AA) modified medical food (PKU formula) and low protein food is necessary on a daily basis for successful PKU management. But as mentioned above, the taste and smell of the AA formulas are offensive, so changing the form of AAs into proteins without Phe would enhance taste, palatability and acceptability of the PKU medical food and ultimately lead to improved dietary compliance.
Glycomacropeptide (GMP), a 64-amino acid glycophosphopeptide cleaved from κ-casein during cheese making, is found in bovine whey[9, 10]. GMP protein is naturally low in Phe, and can be purified further to contain just 2.5-5.0 mg Phe per g of GMP powder[9]. A variety of foods and beverages can be made with GMP to improve the taste, variety and convenience of the PKU diet. It provides a palatable alternative source of protein that may improve dietary compliance and metabolic control of PKU[11, 12]. However, GMP alone does not possess a suitable amino acid profile for PKU treatment and supplement with amino acids including histidine, leucine, tryptophan and tyrosine is therefore required[13]. Therefore, developing a series of recombinant proteins that have suitable amino acid profile as AA formulas and contain low or no Phe will make up for the deficiency of GMP for PKU treatment.
Enzyme Therapy for PKU
There is an increasing interest in enzyme replacement therapy for metabolic diseases. Two enzyme systems are being developed for treatment of PKU: the PAH enzyme and the Phe-degrading enzyme from plants, phenylalanine ammonia-lyase (PAL)[1].
Enzyme Replacement Therapy Using PAH
Enzyme replacement therapy is a viable option to supply active PAH[14]. However, for this to work, there will be a need to administer the PAH cofactor BH4[15], either orally or by addition of the (BH2 to BH4) recycling enzyme dihydropteridine reductase. Although the cofactor requirement is a disadvantage in the use of PAH for enzyme replacement therapy, there are several advantages, which include that the protein is well expressed in bacteria, particularly the doubly truncated form; the expressed protein in the human form of the disease; the protein is easily PEGylated and retain its enzymatic activity, unlike many other enzymes that have been attempted; and the PEGylated protein is very stable after PEGylation. Another advantage of PAH replacement therapy is that additional Tyr supplementation may be unnecessary in PKU therapy. However, the inherent protease sensitivity and potential immunogenicity of PAH have precluded adoption of this approach. Exploring pegylated-PAH as a long-term injectable molecule for PKU is ongoing, but given the drawbacks of the enzyme, its viability as a therapeutic remains debatable[7]. Moreover, using this therapy method high-dose BH4 supplementation is required, which is currently too expensive to afford for most of PKU patients. Therefore, enzyme replacement therapy using PAH will not be a good choice.
Enzyme Replacement Therapy Using PAL
An alternative enzyme therapy for PKU involves the use of PAL, an enzyme capable of substituting for PAH. As a non-mammalian enzyme, PAL is widely distributed in plants, fungi and bacteria. PEgylated PAL derived from algae is currently under investigation for the potential treatment of patients with PKU who do not respond to BH4. PALs can catalyze the conversion of L-phenylalanine to harmless metabolites of trans-cinnamic acid and ammonia without a cofactor requirement. In comparison to PAH, PAL therapy for PKU has some advantages. PAL requires no cofactors for degrading Phe, and trans-cinnamate acid has a very low toxicity and no embryotoxic effects in experimental animals. The PAL product trans-cinnamic acid is converted in the liver to benzoic acid, which is then excreted via the urine mainly as hippurate. PAL is very stable under a wide temperature range.
PAL was investigated to treat PKU as early as 1980 and enzyme replacement therapy studies in human PKU patients began with the oral administration of PAL in entericcoated gelatin capsules. However, when oral administration of the free PALs, enzymes were inactivated rapidly in the gastrointestinal tract due to intestinal proteolysis. Therefore, pretreatment was necessary to protect the PAL enzyme against gastric acidity and pancreatic proteases. Although pharmacological and physiological proofs of principal were attained using PKU mouse model studies, the extreme sensitivity of PAL to low pH and intestinal proteolytic degradation has hindered successful progression of this therapy to clinical trials[14]. However, if an acid stable PAL is found, encapsulated formulation may help reduce plasma Phe. For examples, immobilized PAL within artificial cells was more effective than a phenylalanine-free diet in PKU rats to lower Phe in the plasma, intestinal and cerebrospinal fluids. Oral administration of enteric-coated capsules (ENC) PAL can lower the plasma phenylalanine levels as well. However, oral administration of PAL may need to combine with Phe restrict diet together to get better control of plasma Phe level[19].
Although oral administration of PAL will be more comfortable for the patient, a parenteral modality for PAL therapy needs to be considered. The highly immunogenic property of PAL is a serious problem for parenteral PAL therapy, since it may lead to a short half-life of the enzyme in the blood and unwanted immunologic responses. To overcome these problems, multi-tubular enzyme-reactors with immobilized PAL (from R. glutinis) were investigated and resulted in a rapid, 77% removal of Phe in blood samples of PKU patients[1]. A sustained reduction of Phe was exhibited in less than 1 h, in vitro. Repeated use of PAL reactors in animals did not produce unwanted immunological reactions. However, extracorporeal hollow fibers containing PAL cannot be easily administered to young children, although it may be recommended for PKU management in pregnant women.
Another way to reduce the degree of immunoreactions is PEGylation[1] [16]. The halflives of native PAL and linear PEGylated PAL were 6 and 20 h after the 1st injection, respectively. PEGylated PAL[17] [18] (PEG-PAL, Biomarin Pharmaceuticals) has been shown to suppress immunogenicity and is currently being investigated in Phase 3 clinical trials in the USA. PAL activity is low due to it catalyzes the reversal reaction as well, therefore, a large dose may be required.
Many patents, eg U.S. Pat. No. 5,753,487, EP0260919A1, EP0260919B1, U.S. Pat. No. 4,757,015, EP0703788B1, EP0703788A1, U.S. Pat. No. 4,562,151, U.S. Pat. No. 4,636,466, U.S. Pat. No. 4,681,850, U.S. Pat. No. 4,248,704, U.S. Pat. No. 4,598,047, EP0140707A2, EP0140714A2, U.S. Pat. No. 4,584,273, U.S. Pat. No. 4,584,273, EP0136996A2, JP60172282, JP61139383, JP58086082, U.S. Pat. No. 7,531,341, US 20070048855, U.S. Pat. No. 4,574,117, etc. cover PLA-producing microbial cells, PLA sequence, fermentation, stabilizing agent, variants and chemically-modified variants.
Gene Therapy
Gene therapy for the treatment of PKU has been ongoing over the last 2 decades. The focus has been on replacement of the human mutant PAH gene in somatic cells of PKU patients[20]. Gene therapy is an experimental, yet very promising approach for PKU treatment. Advances in PKU treatment by gene therapy have been accelerated by the availability of pre-clinical models of disease. Early work on gene therapy for children with PKU was considered inappropriate as the therapy involved administration of immunosuppressant agents to block the immune response to the vector so as to prolong the therapeutic effect. Gene Therapy of PKU using viral vectors has had some success in phenotypic correction of the PAHenu2 mice in vivo. Infusion of recombinant adenoviral vectors to the liver resulted in a significant increase in PAH activity leading to complete normalization of the serum Phe levels within one week of treatment. However, the effect did not persist and repeated administrations did not generate the original results due to neutralizing antibodies against the viral vectors. Furthermore, no phenotypic changes were observed and the mice remained hypo-pigmented. In another study, delivery of a recombinant AAV to the liver by portal vein injection resulted in correction of Phe levels in male mice. Females remain unresponsive unless they were ovariectomized and treated with testosterone. The biochemical basis behind this sexual dismorphism was shown to be due to a lower level of BH4 which is controlled primarily by oestrogen and represents a rate limiting factor of PAH activity. Other trials involving the use of recombinant retroviral vectors have been abandoned following the observetion that these vectors may induce leukaemia-like disorders.
Liver-directed gene therapy using recombinant adeno-associated virus serotype 8 vectors (rAAV8) has achieved long-term correction (up to 1 year) of blood Phe concentration in Pahenu2 mice without inducing the immune-mediated rejection seen following adenoviral therapy. However, rAAV8-mediated therapy does not lead to permanent correction of liver PAH deficiency; it is thought that gradual but continuous hepatocyte regeneration eventually leads to elimination of episomal rAAV vector genomes and loss of PAH expression. Reinjection of the same serotype vector is ineffective because of antibody-mediated destruction of the vector.
Initial investigations using non-viral vectors for PKU has thus far been unsuccessful. Injection of naked pDNA by portal vein or hydrodynamic injection with a CMV promoter-driven plasmid resulted in transient PAH expression and a marginal decrease in serum Phe levels, which was not sustained beyond 24 h. Improvements in vector design and engineering using regulatory and/or enhancer elements, as well as insulators, are currently being investigated in order to prolong PAH expression.
Apart from the reports on liver transfection, there are some innovative studies on muscle as a target for gene therapy because adult muscle lacks ongoing cell division. In order to introduce the Phe hydroxylating system into tissue other than the liver, gene delivery must include not only the PAH enzyme but also transport genes that encode the complete enzyme system necessary to synthesize and recycle BH4. Despite such a daunting technical challenge, Ding et al.[21] has shown this to be possible in mice. An advantage of this approach is the ease of access for vectors as compared with liver-directed gene therapy[8].
However, the safety and toxicity and the potential for insertional mutagenesis following viral gene transfer remain an issue. Improvements are necessary to completely eliminate any potential for immune responses. Moreover, after the unfortunate death of a patient with another inborn error of metabolism (ornithine transcarbamylase deficiency)[22], it became clear that there were important issues to be addressed before a gene therapy strategy could be used widely in PKU patients.
BH4 Therapy
The (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is a cofactor in the hydroxylation of Phe to Tyr by PAH[5]. BH4 deficiency accounts for approximately 2% of the high Phe concentrations detected during newborn screening. BH4 is synthesized de novo from GTP by a three enzyme pathway involving GTP cyclohydrolase I (GTPCH I), 6-pyruvoyltetrahydrobiopterin synthase (PTPS) and sepiapterin reductase (SPR) (FIG. 4), and is abundant in the liver. For several decades, BH4 has been used clinically to treat BH4 deficiency, a condition that also can result in elevated blood Phe levels. Several recent studies indicate that approximately 30-50% of PKU patients are responsive to pharmacological doses of BH4, showing a drop in blood Phe levels following a dose of it. However, even some patients with a mutation in the gene encoding PAH, resulting in a clear lack of enzyme activity, might show some response. Experience shows that most of the patients responsive to BH4 still need restriction of natural protein and continued use of low-Phe medical foods. A tablet formulation of BH4 (dihydrochloride) has been available for three decades. Although this formulation has been used extensively in experimental studies, it has not been evaluated in formal clinical trials and was not registered. In addition, BH4 shows limited efficacy in some PKU genotypes and its chemical synthesis is very costly[23].
Many pharmacological chaperones, which are small molecules improving protein stability by rectifying protein folding, have been tried in vitro studies considering PKU as a protein mis-folding disorder[25]. Several researchers have shown that mis-folded PAH protein can be stabilized by BH4 therepy[26, 27]. BH4 prevented the degradation of protein folding variants, proving its effect as a chemical chaperone. A high throughput screening has been performed with more than 1,000 pharmacological compounds and found four compounds which enhanced the thermal stability of wild type PAH and other mutants[28].
A newer formulation of BH4 [sapropterin dihydrochloride (Trade name: Kuvan®), Biomarin Pharmaceuticals] that is more stable at room temperature is now available for the treatment of PKU in the USA and Europe. The synthetic cofactor to PAH, Kuvan® (FIG. 5), acting as a chaperone on the mis-folded PAH protein caused by certain missense PAH mutations, has brought about partial or complete correction of HPA in some patients. Kuvan® is the first drug in the management of PKU approved as an orphan drug by the US Food and Drug Administration (FDA). Sapropterin therapy may also offer relaxation in the strict dietary regimen in patients with mild PKU[29]. Combination of sapropterin with a low-Phe diet increases the stability of blood Phe concentrations and may improve tolerance to dietary Phe[30, 31].
The cost of daily BH4 therapy is very high, for an example, at the highest dose of 20 mg/kg/day, it is US $100,000 to $150,000 for the average adult patient versus the cost of the Phe-restricted diet, including the use of medical foods, which is typically US $15,000 to $20,000 per year. The short half-life (3.3-5.1 h) of BH4 therapy requires frequent dosing, which further accrues treatment cost[5]. Going forward, development of affordable forms of BH4 substitutes or sustained release dosage forms may result in the reduction in the cost of therapy. BH4 supplements may be supplied with classical dietary therapy to achieve better results.
In order to decrease the cost of BH4, some patents have provided the methods for chemical synthesis BH4[33], stable solid formulations BH4[34] and efficiently producing biopterins (BP) by biotransformant[35-38]. The recombinant Escherichia coli show significantly higher productivity, up to 4.0 g of biopterin/L of culture broth[38], which suggests the possibility of commercial BH4 production by biotransformation. There are some patents (WO/2002/018587A1, US20040014167, US20060008869, US20090104668, WO/2006/085535A1, EP1314782A1, EP1314782A4 and CN1449442A) covering the methods for producing BP compounds using BH4 biosynthesis enzymes. Although BP can be bio-transformed by the salvage pathway using BH4 biosynthesis enzyme SPR and DHPR as well, sepiapteriu as the precusor of BH4 biotransformation is also expensive.
Large Neutral Amino Acid Therapy
Large Neutral Amino Acid (LNAA) therapy is an emerging alternative treatment for older individuals with PKU. The concept behind LNAA treatment is that Phe and other LNAA (Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Tyrosine And Valine) share the same transport system, creating competitive inhibition of the transport of LNAA with each other[39, 40]. Therefore, supplementation with LNAA blocks the uptake of Phe by actively controlling cell receptor sites, effectively reducing Phe concentration in the brain. However, the effect of LNAA supplementation on blood Phe concentration might be a result of other factors, including stimulating anabolism or potentially improving the competitive effect of LNAA resulting from decreased natural protein intake, rather than directly influencing transport mechanisms. This is in line with the finding that the blood Phe concentration decreases when amino acid supplements are given more frequently in conjunction with non-LNAA[8].
Supplementation with commercial preparations of LNAA has been noted to reduce brain and circulating levels of Phe in PKU mice and to reduce brain Phe levels in PKU adults off diet as measured by magnetic resonance spectroscopy. LNAAs may be ideal for young adults, for poorly compliant patients, and for late-diagnosed patients in whom compliance is low and in whom drinking formula can be a burden for the patient and caretakers. Adults and older teenagers refusing dietary restrictions can be prescribed a preparation of high-dose LNAAs. The long-term outlook merits further study. Young women of childbearing age need to realize this drug does not protect their fetus from the teratogenic effects of Phe.
Although LNAA treatment does not completely replace the PHA diet, it does help ease the dietary restriction for treated individuals by allowing for a larger amount of natural food protein (FIGS. 6A and B). While diet treatment is always very individual, the LNAA diet may allow the inclusion of regular bread, rice, pasta, and other grains eliminating the need to purchase costly low protein foods. Treatment with LNAA can be especially useful for those struggling with dietary compliance. The ‘relaxed diet’ associated with LNAA therapy, not only improves the dietary options for those living in long-term care environments, but also quality of life. Significant improvements in concentration and decreased self-injurious behavior have been reported in previously untreated adults on a normal diet and LNAA supplementation.
PreKUlab Company has developed some LNAA tablets (PreKUnil and Avonil series, NeoPhe) prekulab.com/products2/neophe-tablets.html). The tablets must be combined with a certain amount of natural protein in order for the diet to contain sufficient protein. After numerous years of experiments and trials with PreKUnil tablets, a group of experts in biochemical and molecular genetics came up with a new formula for PKU treatment—NeoPhe tablets, which has been effective in reducing blood Phe concentrations. However, long-term study of NeoPhe and placebo needs to be conducted in order to establish the efficacy and tolerance of NeoPhe in long term treatment of PKU. Moreover, the taste and smell will also be a problem for people to accept those LNAA tablets. So protein substitute with high in LNAA, but low or no Phe, would control plasma Phe in suitable level, meanwhile enhance taste, palatability and acceptability of the PKU medical food.
As it appears from the description of the various treatment options for PKU, none of the known options provide an optimal solution for the subjects suffering from PKU:
Phe-Restricted Diet with Medical Supplement
The medical food protein that is supplemented to a restricted Phe diet contains amino acids that have an unpleasant taste and smell and it may therefore be difficult to comply with the treatment regime.
Glycomacropeptide (GMP) is not free of Phe and alone it does not possess a suitable amino acid profile for PKU treatment. Thus, the treatment must be supplemented with amino acids like eg histidine, leucine, tryptophanand tyrosine, and, accordingly, the problems relating to taste and smell are not avoided.
The supplement with LNAA e.g. in the form of LNAA tablets as described above also suffers with bad taste and smell and, moreover, the dose is 1 tablet per 1 kg of body weight, which means that e.g. a 60 kg person must intake 60 tablets per day, i.e. 15 tablets 4 times daily or 20 tablets 3 times daily together with a meal. This will most likely also lead to compliance problems.
Enzyme Replacement Therapy
As described herein will enzyme replacement therapy with
i) PAH requires high-dose of BH-4, which is very expensive, and
ii) PAL requires a large dose as its activity is low.
BH4 Therapy
Studies have shown that most of the patients responsive to BH4 still need at least some restriction of natural protein and continued use of low-Phe medical food. Thus, the taste and smell problems are not totally overcome. Moreover, the costs are very high.
As seen from the above, the current treatment options for PKU all have some disadvantages. Accordingly, there is still a need to develop a treatment option of PKU that is without the need for supplement of bad-tasting and bad-smelling amino acids and that is much cheaper than the options relating to enzyme replacement therapy.